Fluorescent labelling of a specific protein of interest (POI) is one of the most widely used methods for studying expression, localization and trafficking of proteins inside living cells. Several labelling techniques have been developed that involve, for example, the use of fluorescent dyes bearing reactive functional groups such as succinimidyl esters or maleimides, known to react with amines or thiols (see, for example, Takaoka, Y. et al., Angew. Chem. Int. Ed. 2013, 52 (15), 4088-4106). However these techniques are typically non-specific, as many such functional groups exposed on the surface of any protein may be labelled, and they do not provide a general means for gathering information on specific protein targets.
Several fluorescent probes for imaging in cell biology have been developed, including small organic dyes, quantum dots, intrinsically fluorescent proteins, small genetically encoded tags that can be complexed with fluorochromes, and combinations of these probes (Giepmans, B. N. et al., Science 2006, 312, 217-24). The most widely applied methods for specific protein labelling include the following: 1) fluorescent protein fusion; 2) small-molecule labelling using protein targeting sequences; 3) enzyme substrate fusion; 4) small-molecule labelling using unnatural amino acids; and 5) small-molecule labelling using peptide targeting sequences. For example, in the first of these methods, an intrinsically fluorescent protein (FP) such as the Aequorea victoria green fluorescent protein (GFP) is genetically fused to a protein of interest (POI). However, there are limitations to this method, including GFP's slow folding, tendency to aggregate, and its steric bulk, all of which can perturb the native biology of a protein of interest. Other methods currently in use all share similar limitations, such as background labelling of native proteins, perturbation of the native biology of a labelled protein of interest, aggregation, attenuation of the fluorescent signal, toxicity, and/or incompatibility for intracellular labelling.
Recently, techniques have been developed to overcome some of the limitations of existing methods through genetic modification of the target protein and subsequent labelling using small molecules. For example, specific amino acids may be introduced into a POI by mutagenesis, or a peptide or protein tag can be fused to the POI's C- or N-terminus, thereby allowing it to react specifically in a covalent or non-covalent manner with a small molecule.
Maleimides are known to react highly selectively with thiols, and have been used especially for labelling peptide thiols. They are also known to be able to quench fluorescence in their conjugated form. At the same time, cysteines are relatively under-represented amino acid residues in naturally occurring proteins, and are often present only in active sites or in tertiary structural motifs as disulfide bonds. Based on these facts, we developed a non-enzymatic small-molecule labelling method, based on the spontaneous, uncatalyzed Fluorogenic Addition Reaction (FlARe) between a fluorogenic labelling agent and a peptide fusion tag. In these methods, as reported previously, a reactive unit bearing two maleimide groups is linked to a fluorophore, such that fluorescence is quenched by photoinduced electron transfer (PET) until both maleimide groups undergo specific thiol addition reactions (see, e.g., U.S. Pat. No. 7,700,375; U.S. Pat. No. 8,835,641; U.S. Patent Application Publication No. 2015-0316557; Caron, K. et al., Org. Biomol. Chem. 9, 185-197, 2011; Guy, J. et al., Mol. Biosyst. 6: 976-987, 2010; Keillor, J. W. et al., J. Am. Chem. Soc. 129, 11969-11977, 2007; Girouard, S. et al., J. Am. Chem. Soc. 127, 559-566, 2005).
A complementary alpha-helical peptide tag was also designed for use in FlARe labelling methods. Specifically, we synthesized a dicysteine peptide tag bearing two cysteine residues whose thiol side chains are appropriately positioned to react specifically with the two maleimide groups of a fluorogenic molecule comprising a dimaleimide moiety and a fluorophore (U.S. Pat. No. 8,835,641; Guy, J. et al., Mol Biosyst 2010, 6, 976-987). This dicysteine tag (referred to as dC10, for a di-Cysteine alpha-helix with an inter-Cys distance of ˜10 Å) was designed to include salt bridges that lock the peptide in a helical conformation and ensure aqueous solubility, as well as a C-cap and an N-cap that confer stability to the small helix by preserving its dipole. Further, the two cysteine residues in the dC10 peptide were separated by two turns of the alpha-helix, and therefore by ˜10 Å, to complement fluorogen molecules whose reactive maleimide moieties are also ˜10 Å apart.
Genetically fusing these helical peptides to test proteins of interest (POI), we were able to selectively label the target sequence. However, although the dC10 tag has proven useful, the labelling methods are limited by the reactivity and selectivity of the peptide tag.
There is a need therefore for improved peptide tags that are more reactive and/or selective for use in FlARe fluorescent labelling methods.